The lab-grown brain, about the size of a pencil eraser, has an identifiable structure and contains 99 percent of the genes present in the human foetal brain. Such a system will enable ethical and more rapid and accurate testing of experimental drugs before the clinical trial stage and advance studies of genetic and environmental causes of central nervous system disorders.

“It not only looks like the developing brain, its diverse cell types express nearly all genes like a brain,” Anand said.

“We’ve struggled for a long time trying to solve complex brain disease problems that cause tremendous pain and suffering. The power of this brain model bodes very well for human health because it gives us better and more relevant options to test and develop therapeutics other than rodents.”

Anand reported on his lab-grown brain Tuesday (Aug. 18) at the 2015 Military Health System Research Symposium in Ft. Lauderdale, Florida.

This image of the lab-grown brain is labelled
to

show identifiable structures: the cerebral

hemisphere, the optic stalk and the cephalic

flexure, a bend in the mid-brain region, all

characteristic of the human foetal brain. Credit:

courtesy of The Ohio State University.

Anand, who studies the association between nicotinic receptors and central nervous system disorders, was inspired to pursue a model of human neural biology after encountering disappointing results in a rodent study of an experimental autism drug. Taking a chance with a shoestring budget compared to other researchers doing similar projects, he added stem-cell engineering to his research program. Four years later, he had built himself a replica of the human brain.

The main thing missing in this model is a vascular system. What is there – a spinal cord, all major regions of the brain, multiple cell types, signalling circuitry and even a retina – has the potential to dramatically accelerate the pace of neuroscience research, said Anand, also a professor of neuroscience.

“In central nervous system diseases, this will enable studies of either underlying genetic susceptibility or purely environmental influences, or a combination,” he said.

“Genomic science infers there are up to 600 genes that give rise to autism, but we are stuck there. Mathematical correlations and statistical methods are insufficient to in themselves identify causation. You need an experimental system – you need a human brain.”

Converting adult skin cells into pluripotent cells – immature stem cells that can be programmed to become any tissue in the body – is a rapidly developing area of science that earned the researcher who discovered the technique, Shinya Yamanaka, a Nobel Prize in 2012.

“Once a cell is in that pluripotent state, it can become any organ – if you know what to do to support it to become that organ,” Anand said.

“The brain has been the holy grail because of its enormous complexity compared to any other organ. Other groups are attempting to do this as well.”

Anand’s method is proprietary and he has filed an invention disclosure with the university.

He said he used techniques to differentiate pluripotent stem cells into cells that are designed to become neural tissue, components of the central nervous system or other brain regions.

“We provide the best possible environment and conditions that replicate what’s going on in utero to support the brain,” he said of the work he completed with colleague Susan McKay, a research associate in biological chemistry and pharmacology.

High-resolution imaging of the organoid identifies functioning neurons and their signal-carrying extensions – axons and dendrites – as well as astrocytes, oligodendrocytes and microglia. The model also activates markers for cells that have the classic excitatory and inhibitory functions in the brain, and that enable chemical signals to travel throughout the structure.

It takes about 15 weeks to build a model system developed to match the 5-week-old foetal human brain. Anand and McKay have let the model continue to grow to the 12-week point, observing expected maturation changes along the way.

“If we let it go to 16 or 20 weeks, that might complete it, filling in that 1 percent of missing genes. We don’t know yet,” he said.

He and McKay have already used the platform to launch their own projects, creating brain organoid models of Alzheimer’s and Parkinson’s diseases and autism in a dish. They hope that with further development and the addition of a pumping blood supply, the model could be used for stroke therapy studies. For military purposes, the system offers a new platform for the study of Gulf War illness, traumatic brain injury and post-traumatic stress disorder.

Support for the work came from the Marci and Bill Ingram Research Fund for Autism Spectrum Disorders and the Ohio State University Wexner Medical Center Research Fund.

Anand and McKay are co-founders of a Columbus-based start-up company, NeurXstem, to commercialize the brain organoid platform, and have applied for funding from the federal Small Business Technology Transfer program to accelerate its drug discovery applications.

Friday, 7 August 2015

Two labs in China have independently succeeded in transforming skin cells into neurons using only a cocktail of chemicals, with one group using human cells from healthy individuals and Alzheimer's patients, and the other group using cells from mice. The two studies reinforce the idea that a purely chemical approach is a promising way to scale up cell reprogramming research that may avoid the technical challenges and safety concerns associated with the more popular method of using transcription factors. Both papers appear on August 6 in the journal Cell Stem Cell.

One of the challenges of forcing cells to change identity is that the cells you end up with may look normal but have different internal activities than their naturally forming counterparts. The two papers provide evidence that similar gene expression, action potentials, and synapse formation can be detected in transcription-factor-induced neurons as those generated from the chemical cocktails. (Both groups used mixtures of seven small molecules, but different recipes – outlined in detail in the supplemental information section of each paper – because they focused on different species.)

This is
an image of mouse chemical-induced

neurons.
Credit: Courtesy of Hongkui Deng.

"We found that the conversion process induced by our chemical strategy is accompanied by the down-regulation of [skin-cell] specific genes and the increased expression of neuronal transcription factors," said human study co-author Jian Zhao, of the Shanghai Institutes for Biological Sciences and Tongji University.

The authors add that the direct conversion bypasses a proliferative intermediate progenitor stage, which circumvents safety issues posed by other reprogramming methods.

This is
an image of human chemical induced

neurons.
Credit: Courtesy of Gang Pei and

Jian
Zhao.

Zhao's paper, co-led with cell biologist Gang Pei, also shows that the pure chemical protocol can be used to make neurons from the skins cells of Alzheimer's patients. Most of the work using patient stem cells has been done by using transcription factors – molecules that affect which genes are expressed in a cell – to create induced pluripotent stem cells. Chemical cell reprogramming is seen as an alternative for disease modelling or even potential cell replacement therapy of neurological disorders, but the "proof-of-concept" is still emerging.

"In comparison with using transgenic reprogramming factors, the small molecules that are used in this chemical approach are cell permeable; cost-effective; and easy to synthesize, preserve, and standardize; and their effects can be reversible," says mouse study co-author Hongkui Deng of the Peking University Stem Cell Research Center.

"In addition, the use of small molecules can be fine-tuned by adjusting their concentrations and duration, and the approach bypasses the technical challenges and safety concerns of genetic manipulations, which may be promising in their future applications."

Deng worked for four years with Zhen Chai and Yang Zhao, also of Peking University, to identify the small molecules that could create chemically induced mouse neurons. Researchers had been close for years, but a transcription factor was always necessary to complete the transformation. Through many chemical screens they identified the key ingredient, I-BET151, which works to suppress transcription in skin cells. They then found the right steps and conditions to mature the neurons post-transformation.

The authors of both papers aim to learn more about the biology behind chemically induced reprogramming and to make the protocols more efficient. While their success is promising, there are still a number of hurdles to overcome.

"We hope in the future that the chemical approaches would be more robust in inducing functional mature neurons," Deng says.

"In addition, we are attempting to generate specific neuronal subtypes and patient-specific functional neurons for translational medicine by using pure chemicals."

Jian Zhao, of the human study, says:

"It should be possible to generate different subtypes of neurons with a similar chemical approach but using slightly modified chemical cocktails."

"It also needs to be explored whether functional neurons could be induced by chemical cocktails in living organisms with neurological diseases or injury," she adds.

Just after fertilization, when the embryo is comprised of only 1 or 2 cells, cells are "totipotent", that is to say, capable of producing an entire embryo as well as the placenta and umbilical cord that accompany it. During the subsequent rounds of cell division, cells rapidly lose this plasticity and become "pluripotent". At the blastocyst stage (about thirty cells), the so-called "embryonic stem cells" can differentiate into any tissue, although they alone cannot give birth to a foetus anymore. Pluripotent cells then continue to specialise and form the various tissues of the body through a process called cellular differentiation.

For some years, it has been possible to re-programme differentiated cells into pluripotent ones, but not into totipotent cells. Now, the team of Maria-Elena Torres-Padilla has studied the characteristics of totipotent cells of the embryo and found factors capable of inducing a totipotent-like state.

“Totipotency is a much more flexible state than the pluripotent state and its potential applications are extraordinary”, says Maria-Elena Torres-Padilla, who led the study.

Looking for the keys of totipotency

When culturing pluripotent stem cells in vitro, a small amount of totipotent cells appear spontaneously; these are called "2C-like cells" (named after their resemblance to the 2-cell stage embryo). The researchers compared these cells to those present in early embryos in order to find their common characteristics and those that make them different from pluripotent cells. In particular, the teams found that the DNA was less condensed in totipotent cells and that the amount of the protein complex CAF1 was diminished. A closer look revealed that CAF1 – already known for its role in the assembly of chromatin (the organised state of DNA) – is responsible for maintaining the pluripotent state by ensuring that the DNA is wrapped around histones. Based on this hypothesis, the Torres-Padilla team was able to induce a totipotent state by inactivating the expression of the CAF1 complex, which led to chromatin reprogramming into a less condensed state.

A 2C-like cell (green) is different from an

embryonic stem cell (magenta). Credit:

IGBMC/Maria-Elena Torres-Padilla.

In order to carefully examine at a molecular level the similarities between 2-cell stage embryos, 2C-like cells and those induced by inactivating the CAF1 complex, the Torres-Padilla team then joined forces with the Vaquerizas laboratory to analyse, in a genome-wide fashion, the gene expression programmes of these cells. The scientists found that the induced, CAF1-depleted, totipotent cells overexpressed a significant amount of 2-cell stage embryo genes.

“One could imagine that if cells lose their ability to assemble chromatin, this would affect gene expression”, explains Cells-in-Motion PhD student Rocio Enriquez-Gasca of Juanma Vaquerizas’ lab, who performed the computational analyses of the work.

“So it was really exciting to realise that the resulting gene expression programme in fact significantly overlaps with that of early embryo, totipotent cells”.

Moreover, the teams found that specific classes of repetitive elements (repeated sequences of DNA that form around 50% of the mouse and human genomes) were also up-regulated in induced totipotent-like cells, a hallmark of the 2-cell embryo.

“The computational analysis of expression of repetitive elements is very challenging, since these are found many times in the genome”, says Juanma Vaquerizas.

“Now it is key to understand why these repetitive elements and gene expression programmes are both up-regulated in totipotent cells”.

These results provide new elements for the understanding of pluripotency and could increase the efficiency of reprogramming somatic cells to be used for applications in regenerative medicine.